: The vesicular acetylcholine transporter (VAChT) stores acetylcholine (ACh) for release from the nerve terminal. A structure-function analysis of VAChT will be done by site-directed mutagenesis, expression and characterization of changes in transporter, inhibitor binding, pH dependence and ACh washout properties of the mutants. A microscopic kinetics model of the VAChT transport cycle will be important to interpretation. The sequences of related vesicular monoamine transporter (VMAT) and bacterial multidrug resistance (mdr) transporters will guide strategy. The goal is to assign specific amino acids to specific functions. Aim 1 seeks to identify the second of the two proton-translocation sites. The possibility that the core proton-translocation mechanism is not conserved between VAChT and VMAT will be tested in transport-rescue experiments. Aim 2 seeks to identify the residue of pKa 7.1 that must be deprotonated to bind ACh and the residue of pKa > 9 that must be protonated to transport bound ACh. Aim 3 seeks to localize the ACh binding site. Also, possible electrostatic guidance for ACh binding will be investigated. Aim 4 will test whether any of six conserved conformational motifs first identified in bacterial mdr transporters control conformational changes in the VAChT transport cycle. Conserved residues flanking the motifs also will be tested for conformational roles. The project will identify features of VAChT mediating or controlling transport, including those potentially limiting to ACh storage. This might reveal a suitable target for development of a drug that increases ACh storage by modulating VAChT function. The long term goal is effective treatment of cholinergic hypofunction.